Project Summary/Abstract PCSK9 downregulates the function of the LDL receptor, which raises LDL cholesterol in the bloodstream and accelerates atherosclerotic heart disease. PCSK9 interacts with the LDL receptor on the cell surface, and in order to do so, PCSK9 must first be successfully secreted. Current antibody-based therapies against PCSK9 work well, lowering LDL cholesterol and reduce the incidence of heart attack, but are expensive and not-cost effective. To address this problem, we propose to investigate the biology of PCSK9 secretion, as this may serve as an alternative approach to target PCSK9-based regulation of the LDL receptor. Based on work by ourselves and others, we hypothesize that PCSK9 secretion follows a highly regulated pathway that requires the assistance of several other proteins. In Aim 1, we will investigate the relationship of the p24 family of proteins to PCSK9 secretion. Using a combination of small molecule inhibitors, gene overexpression, and CRISPR mediated gene knockdown, we will as whether this family of proteins acts to recognize properly folded PCSK9 and permit forward movement in the secretory pathway, or if instead this family acts to restrict and retain improperly folded PCSK9. In Aim 2, we will search for genetic mediators required for PCSK9 secretion and for the interaction between PCSK9 and SEC24A, a component previously known to be required for PCSK9 secretion. We will achieve this through a combination of co-immunoprecipitation, proximity-based biotinylation, and genome-wide CRISPR screens. The results of our study will 1) generate new tools to study PCSK9 secretion, 2) determine whether modulating PCSK9 secretion is a viable therapeutic strategy, 3) discover potential genetic targets against atherosclerosis, and 4) open up new areas of PCSK9 biology. Because PCSK9 has such a critical role in regulating serum LDL cholesterol, these results will have substantial clinical relevance.